Conditionally active chimeric antigen receptors for modified T-cells
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Publication Number
US-11584927-B2
Publication Date
2023-02-21
Expiration Date
Abstract
This disclosure relates to a chimeric antigen receptor for binding with a target antigen. The chimeric antigen receptor comprises at least one antigen specific targeting region including a multispecific antibody evolved from a wild-type antibody or a fragment thereof and having at least one of: (a) a decrease in activity in the assay at the normal physiological condition compared to the wild-type antibody or the fragment thereof, and (b) an increase in activity in the assay under the aberrant condition compared to the wild-type antibody or the fragment thereof. A method for using the chimeric antigen receptor and cytotoxic cells for cancer treatment is also provided. A method for producing the chimeric antigen receptor is also provided.
Core Innovation
The invention relates to conditionally active biologic proteins and conditionally active antibodies that are less active in a normal physiological environment but more active in a disease microenvironment characterized by an aberrant pH, including a tumor microenvironment. It addresses avoiding collateral damage and reducing systemic toxicity while maintaining activity where aberrant pH is present.
The approach uses selection criteria based on binding affinity ratios between tumor and non-tumor microenvironments, including conditions with aberrant acidic pH and normal physiological pH. It extends the conditional-activity concept to targeting stem cell niches, including cancer stem cells, inflammation-susceptible organs or tissues, and conditional delivery or recognition proteins presented on viral particles.
The document further describes conditionally active genome-editing Cas proteins having pH-dependent enzymatic activity with engineered conditionally active antigen-specific targeting regions linked to CAR assembly and use within CAR-T cell platforms. Illustrative embodiments include pH-selective conditionally active scFv antibodies selected for selectivity between pH 6.0 and 7.4, and CAR-T cell activity characterized by cytotoxicity and cytokine release against antigen-expressing target cells with reduced activity toward antigen-negative controls.
Claims Coverage
The document provides one independent claim focused on producing a CAR by evolving an antigen-binding region and selecting a conditionally active region based on decreased binding under normal physiological pH relative to aberrant pH, then linking the selected region into a CAR comprising a transmembrane domain and an intracellular signaling domain.
Evolving an antigen specific targeting region from a parent protein
Generating the at least one antigen specific targeting region from a parent protein or a domain thereof that binds specifically with the target antigen by evolving the DNA encoding the parent protein or a domain thereof to create mutant DNAs and expressing the mutant DNAs to obtain mutant polypeptides.
Selecting conditionally active binding by normal versus aberrant pH assay
Subjecting the mutant polypeptides to an assay under a normal physiological pH and to an assay under an aberrant pH below the normal physiological pH, and selecting a conditionally active antigen specific targeting region that exhibits a decrease in binding activity at the normal physiological pH compared to binding activity under the aberrant pH.
Linking the selected targeting region to CAR transmembrane and intracellular signaling domains
Linking the selected conditionally active antigen specific targeting region to the transmembrane domain and the intracellular signaling domain.
Overall, the claim coverage centers on conditioning antigen-specific binding through pH-dependent selection of evolved targeting regions, and on integrating the selected conditionally active targeting region into a CAR architecture with a transmembrane domain and an intracellular signaling domain.
Stated Advantages
Reduced systemic toxicity while maintaining targeted activity in disease microenvironments characterized by aberrant pH.
Reduced collateral damage.
Documented Applications
Immune checkpoint inhibition targeting immune checkpoint proteins such as CTLA-4 and PD-1, using conditionally active biologic proteins or antibodies in disease microenvironments with aberrant pH.
Targeting cancer stem cells and stem cell niches using conditionally active targeting concepts based on disease microenvironment conditions.
Targeting inflammation-susceptible organs or tissues using conditionally active targeting concepts based on disease microenvironment conditions.
Conditional delivery or recognition proteins presented on viral particles.
Conditionally active genome-editing using pH-dependent enzymatic activity Cas proteins.
CAR-T applications in which pH-selective conditionally active scFv antibodies and CAR assembly are used, with characterization including cytotoxicity and cytokine release against antigen-expressing target cells.
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